NMDA receptors are membrane-bound neurotransmitter receptors with critical roles in brain physiology. They are principal drug targets for Alzheimer's disease and stroke, mental retardation, and also addiction, chronic pain, schizophrenia and epilepsy. NMDA receptors are glutamate-activated, excitatory ion-channels. Their activation reaction is initiated when the neurotransmitter glutamate binds to extracellular, ligand-binding domains (LBDs) and culminates with the opening of a membrane-embedded cation-selective pore. The molecular events that make up the activation reaction of NMDA receptors remain unknown, despite their fundamental role in controlling receptor function. Kinetic studies of single-receptor activity have established that after binding agonists, NMDA receptors cycle with measurable rates between several closed-pore and open-pore conformations. Structural studies of the isolated LBDs have shown that ligands bind deep inside a crevice between two mobile lobes and have put forth the hypothesis that ligand-induced closure of the LBD is one of the required steps along the NMDA receptor activation pathway. This application proposes experiments that will directly and specifically test this hypothesis by examining the activity of NMDA receptors with restricted mobility of the LBDs. Pairs of cysteine residues will be introduced at the tips of the LBDs lobes as a means to lock these at defined distances with respect to one another. The engineered receptors will be examined by kinetic analyses of their macroscopic and single-channel behaviors in the presence and absence of oxidizing agents, and following treatment with crosslinking reagents of various lengths. The results will provide basic information regarding the identity of the molecular motions associated with NMDA receptor activation and will afford valuable insight regarding possible mechanisms of drug action at this receptor. This knowledge will form the conceptual foundation necessary to rationally control NMDA receptor activities as a therapeutic strategy for stroke, addiction, and mental illness. PUBLIC HEALTH RELEVANCE: This project will provide fundamental information regarding the mechanism by which NMDA receptors become active. This information is currently lacking and is needed for the rational design of novel therapeutic approaches for acute and chronic neurodegeneration (stroke, Alzheimer's disease), addiction, chronic pain, cognitive disorders and mental illness.